Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)

  Western blot - pan methyl Lysine (methyl K pan) antibody - ChIP Grade (ab7315)

All lanes : Anti-pan methyl Lysine antibody - ChIP Grade (ab7315) at 1/500 dilution

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 1 µg/ml
Lane 3 : Calf thymus histone lysate with Histone H3 peptide - di methyl K4 (ab7768) at 1 µg/ml
Lane 4 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 1 µg/ml
Lane 5 : Calf thymus histone lysate with Histone H3 peptide - unmodified (ab2903) at 1 µg/ml
Lane 6 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 1 µg/ml
Lane 7 : Calf thymus histone lysate with Histone H3 peptide - di methyl K27 (ab1781) at 1 µg/ml
Lane 8 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K27 (ab1782) at 1 µg/ml
Lane 9 : Calf thymus histone lysate with Histone H3 peptide - unmodified (ab2623) at 1 µg/ml
Lane 10 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 1 µg/ml
Lane 11 : Calf thymus histone lysate with Histone H3 peptide - di methyl K9 (ab1772) at 1 µg/ml
Lane 12 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 1 µg/ml

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size : 14-20 kDa


Exposure time : 5 minutes

Expected molecular weights: H3 = 20 kDa; H4 = 14 kDa

This image shows that the main epitopes recognized by ab7315 are the di methylated lysine residues. This can be seen in lanes 3, 7 and 11 where the activity of the antibody is quenched by the immunizing peptides (ab7768, ab1781, ab1772). The antibody also recognises mono and tri methylated residues, but to a lesser extent.

  ChIP - pan methyl Lysine (methyl K pan) antibody - ChIP Grade (ab7315)

 Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 6.5µl of ab7315 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - pan methyl Lysine (methyl K pan) antibody - ChIP Grade (ab7315)

IHC image of pan methyl Lysine (methyl K pan) staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7315, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.